a. Field
The present disclosure relates generally to compositions and methods for manufacture and use of a multiple strain containing microbial product. More specifically, the disclosure relates to methods for improving animal health and/or productivity through the use of probiotic microorganisms.
b. Description of the Related Art
One of the larger economic burdens facing dairy farmers is the high cost of rearing and/or replacing heifers to maintain or increase herd size. A major factor contributing to the high cost of heifer replacement is the prevalence of diarrheal disease, known as scours, in livestock. Scours causes greater than 60% of all deaths associated with pre-weaned calves, and accounts for 6.2% of total calf losses. The prevalence of scours can vary dramatically (4.3% to 52.4%) depending on herd, diet, season, or “outbreak” occurrences. Estimates of scouring rates within a herd are difficult to obtain, though they are believed to between 15% and 35%. Nonetheless, it is agreed that diarrheal events comprise the largest health challenge to pre-weaned calves.
Diarrhea (scours) remains the predominant cause of mortality among dairy calves. There are multiple causes of scours including malabsorption and improper nutrition; however, infections by bacteria, viruses, and protozoa are the primary etiological agents. It is important to consider that scours in calves may be due to a number of concurrent gastrointestinal insults by numerous pathogens. Susceptibility to acute undifferentiated diarrhea can be largely determined by the quantity, quality, and administration time of colostrum.
The costs associated with scours are difficult to estimate; however, mortality alone represents a large expense, since, at birth, a heifer has an estimated value of $400-$600. Scours does not always result in death, but costs associated with treatment (e.g. electrolytes, antibiotics, veterinary services and associated labor) can be significant. In addition, animal sickness and death can negatively impact the morale of farm laborers and must be taken into consideration, though the financial costs of this cannot be readily quantified.
Serum immunoglobulin obtained from colostrum can offer some limited protection to calves from bacterial and viral infections. However, this protective effect begins to diminish <96 hours after birth, which could explain the high onset of viral scours 5-7 days following birth (Radostits 2000). Prophylactic antibiotics and vaccines administered to calves are frequent measures used to prevent scours in calves. While antibiotic administration can be effective against bacterial infections, antibiotics are ineffective against viruses and protozoa and, in fact, they can promote the development of viral or protozoal scours by diminishing the normal protective flora. Vaccinations can also confer protection against scours, however, the full protective immune response does not occur until after few weeks of administration. Despite some advances in prevention and treatment, the incidence of scours can vary wildly between dairy herds.
A major factor contributing to the onset of scours in calves is the practice of removing calves from their mother cows immediately after birth, and transporting them to facilities away from adult animals. The gastrointestinal tracts of mammals, including calves, are sterile at birth, but rapidly become colonized by microflora located near the mother's vagina and anus. Other bacteria begin to establish themselves when the neonate comes into contact with new objects (feed, dirt, gates, fences, handlers, etc.). Prior to the current practice of removing a calf from its mother, protective microflora would become established in the calf due to contact with the mother via licking, nursing, and grooming. Thus, one possible avenue to reduce the incidence and severity of scours includes manipulating the microbial flora of a calves' digestive tract.
It has long been known that a number of beneficial bacteria colonize the intestinal tracts of mammals and can promote the well being of the host. It has also been recognized for many years that the consumption of exogenous bacteria, often referred to as probiotics, can elicit beneficial effects upon a host. In humans, these probiotic bacteria have been shown to reduce the severity and duration of rotaviral-induced diarrhea, alleviate lactose intolerance, and enhance gastrointestinal immune function (Roberfroid 2000). Traditionally, food sources such as yogurt have been considered probiotic-carriers providing these health-promoting benefits. It is believed that the consumption of foods rich in probiotic bacteria, including lactic acid bacteria and bifidobacteria, leads to colonization of the human gastrointestinal tract of humans (Roberfroid 2000).
It is also well established that the addition of probiotic microorganisms to animal feed can improve animal efficiency and health. Specific examples include increased weight gain-to-feed intake ratio (feed efficiency), improved average daily weight gain, improved milk yield, and improved milk composition by dairy cows as described by U.S. Pat. Nos. 5,529,793 and 5,534,271. The administration of probiotic organisms can also reduce the incidence of pathogenic organisms in cattle, as reported by U.S. Pat. No. 7,063,836.
Researchers have demonstrated that the consumption of probiotics by animals used in food production can improve the efficiency of animal production. Probiotics may work by competitive exclusion in which live microbial cultures act antagonistically on specific organisms to cause a decrease in the numbers of that organism. U.S. Pat. No. 7,323,166. Mechanisms of competitive exclusion include production of antibacterial agents (bacteriocins) and metabolites (organic acids and hydrogen peroxide), competition for nutrients, and competition for adhesion sites on the gut epithelial surface. U.S. Pat. No. 7,323,166. Lactic acid bacteria are generally considered as food grade organisms and there are many potential applications of protective cultures in various foods. A number of different factors have been identified that contribute to the antimicrobial activity of lactic acid bacteria. These bacteria produce different antimicrobials, such as lactic acid, acetic acid, hydrogen peroxide, carbon dioxide and bacteriocins, which can inhibit pathogenic microorganisms.
Propionic acid is important in ruminal and intestinal fermentations and is a precursor to blood glucose synthesis (Baldwin 1983). Several examples are available that demonstrate the positive impact of feeding propionic acid-producing organisms to cattle. For example, U.S. Pat. Nos. 5,529,793 and 5,534,271, 6,455,063 and 6,887,489 demonstrate beneficial effects of propionic acid-producing bacteria upon cattle growth. Lactic acid bacteria (LAB) can inhibit pathogens in various food sources (Brashears et al., 2003). Lactic acid producing and lactate utilizing bacteria may also be helpful in inhibiting pathogenic growth in animals and improving the production of dairy products. U.S. Pat. No. 7,063,836. Lactic acid producing and lactate utilizing bacteria are beneficial for the utilization of feedstuffs by ruminants (U.S. Pat. Nos. 5,529,793 and 5,534,271) and have been fed to cattle to improve animal performance (Brashears et al., 2003).
Propionic acid is important in ruminal and intestinal fermentations and is a precursor to blood glucose synthesis (Baldwin 1983). Several examples are available that demonstrate the positive impact of feeding propionic acid-producing organisms to cattle. For example, U.S. Pat. Nos. 5,529,793 and 5,534,271, issued to Garner and Ware, along with U.S. Pat. Nos. 6,455,063 and 6,887,489, issued to Rehberger et al., teach of the beneficial effects that propionic acid-producing bacteria have upon cattle growth. Lactic acid bacteria (LAB) can inhibit pathogens in various food sources. Brashears et al., 2003. Lactic acid producing and lactate utilizing bacteria may also be helpful in inhibiting pathogenic growth in animals and improving the production of dairy products. U.S. Pat. No. 7,063,836. Lactic acid producing and lactate utilizing bacteria are beneficial for the utilization of feedstuffs by ruminants (U.S. Pat. Nos. 5,529,793 and 5,534,271) and have been fed to cattle to improve animal performance. Brashears et al., 2003.
Bacteriocins are ribosomally synthesized extracellularly released bioactive peptides or peptide complexes which have bacteriocidal or bacteriostatic activity. The producer cells exhibit immunity to the action of its own bacteriocin. Bacteriocin producing strains can be identified in a deferred antagonism assay where colonies of putative producer cells are covered with a bacterial strain which is sensitive to the bacteriocins. After incubation, zones of inhibition are visible. Bacteriocins are known to inhibit foot borne pathogens such as Clostridium botulinum, Enterococcus faecalis, Listeria monocytogenes and Staphylococcus aureus. 
Four general classes of bacteriocins have been characterized: 1) lantibiotics, 2) small <13 kDa hydrophobic heat stable peptides, 3) large >30 kDa heat labile proteins and 4) complex proteins that require additional carbohydrate or lipid moieties to attain antimicrobial activity. Lantibiotics are a family of membrane active peptides that contain a thio-ether amino acid known as lanthionine and β-methyl lanthionine as well as other modified amino acids such as dehydrated serine and threonine. A particular feature of lantibiotics is the presence of post translationally modified amino acid residues. One example of a lantibiotic is nisin. Bacteriocins which are small heat stable peptides do not contain modified amino acid residues. Large heat labile bacteriocins include helviticin-J and lactacins A and B.
A majority of bacteriocins produced by bacteria are lantibiotics or small hydrophobic heat stable peptides. Nisin, a lantibiotic is effective at inhibition of Gram-positive bacteria such as Bacillus and Clostridium. However, Nisin has demonstrated no effectiveness against Gram-negative bacteria. Among the small hydrophobic heat stable peptides, pediocins are frequently encountered and possess the ability to inhibit Listeria monocytogenes. 
Lactobacillus genus includes the most prevalently administered probiotic bacteria (Flint and Angert 2005). Lactobacillus is a genus of more than 25 species of gram-positive, catalase-negative, non-sporulating, rod-shaped organisms (Heilig et al., 2002). Lactobacillus species ferment carbohydrates to form lactic acid as reported in U.S. Pat. No. 7,323,166. Lactobacillus species are generally anaerobic, non-motile, and do not reduce nitrate as reported in U.S. Pat. No. 7,323,166. Lactobacillus species are often used in the manufacture of food products including dairy products and other fermented foods as reported by Heilig et al., 2002 and U.S. Pat. No. 7,323,166. Lactobacillus species inhabit various locations including the gastrointestinal tracts of animals and intact and rotting plant material as reported by Heilig et al., 2002 and U.S. Pat. No. 7,323,166. Lactobacillus strains appear to be present in the gastrointestinal tract of approximately 70% of humans that consume a Western-like diet. Heilig et al., 2002. The number of Lactobacillus cells in neonates is approximately 105 colony forming units (CFU) per gram CFU/g of feces. Heilig et al., 2002. The amount in infants of one month and older is higher, ranging from 106 to 108 CFU/g of feces. Heilig et al., 2002.
Lactic acid and products containing lactic acid have been found to enhance gains in the starting period of cattle (first 28 days) and reduce liver abscesses when given prior to the transition from a roughage diet to a feedlot diet. Various strains of Lactobacillus acidophilus have been isolated which restore and stabilize the internal microbial balance of animals. Manfredi et al., U.S. Pat. No. 4,980,164, is such a strain of Lactobacillus acidophilus which has been isolated for enhancing feed conversion efficiency. The Lactobacillus acidophilus strain of the Manfredi et al patent, has been designated strain BT1386 and received accession number ATCC No. 53545 from the American Type Culture Collection in Rockville, Md. Strain ATCC 53545 demonstrates a greater propensity to adhere to the epithelial cells of some animals which would increase their ability to survive, initiate and maintain a population within an animal intestine. Thus, the primary mode of action as previously understood relative to Lactobacillus acidophilus occurs post-ruminally.
The most common method used today to control pathogenic populations in livestock is through the use of antimicrobial compounds. While these are effective for short-term treatments, prolonged application of antimicrobial compounds leads to the evolution of antibiotic resistance in pathogenic organisms. The widespread occurrence of antibiotic resistant microorganisms is well known, some of the most common being methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE). Bacteria are remarkably adaptable to deleterious environments with their abilities to rapidly reproduce and modify their genetic content. Thus, it is inevitable that after prolonged application of any method that disrupts or kills bacteria a population that is recalcitrant to its effects will eventually arise. It is not uncommon now in the medical environment that doctors often resort to using multiple antibiotics concurrently or in succession to eradicate pathogenic organisms.
As with antibiotics, bacteria can also become resistant to other biological treatments. For example, bacteriophages are able to reduce pathogen populations, but inevitably, a fraction of the targeted bacterial population is not infected. This small sub-population then rapidly reproduces and attains sizable population numbers. Some researchers have been able to overcome this by using multiple phages in a “cocktail” to reduce pathogen populations further than if only one was used. The premise behind including multiple phage is that different phage utilize different sites for attachment and infection of the host bacterium. While a cell can become resistant to one phage by modifying the phage attachment site, it is more difficult for the bacterium to modify two, three, or more attachment loci to evade all of the different phage in the cocktail.
Similar circumstances have been seen with the application of probiotic bacteria that are meant to inhibit or reduce the numbers of pathogenic bacteria within a gastrointestinal system. Some researchers have commented that significantly better animal performance and pathogen reductions were seen in treated animals early in their experiments, but the beneficial effects were no longer statistically different after prolonged application of the probiotic product. It is possible that the target populations were initially affected, but prolonged usage of the probiotic product led to the selection of bacterial populations that were not influenced by the application of the product. However, as seen with multiple phage application, it may be possible to avoid the adaptation of pathogens to probiotic treatment with the inclusion of multiple strains of bacteria.
There are numerous advantages for the inclusion of multiple strains of microorganisms in a microbial product. The potential advantages described below, whether working independently or concurrently, allow for a superior microbial product and enhanced benefits for the host.
Different microbial strains utilize certain nutrients more efficiently than others. The ability to use available nutrients in a gut environment is necessary for the microbe to produce antimicrobial compounds or to beneficially affect the host GI system. However, the nutrient availability is constantly changing because of animal behavior, different foods consumed, antibiotic use, energy requirements, or health of the animal. These fluctuations allow for different microbes to proliferate while other microbial populations diminish.
The use of different microbial strains also allows for the production of different microbial metabolites. Different metabolites have different effects upon pathogenic populations. Lactic acid is a powerful antimicrobial agent against some pathogens, while propionic acid is more effective against other populations. It should also be considered that just as metabolites produced from cells from the microbial product affect GI populations, endogenous microorganisms produce chemicals that may be inhibitory to some strains in the microbial product. The inclusion of different strains in a product increases the likelihood that the product will have a positive effect.
Additionally, the production of bacteriocins is known to influence bacterial populations. There is a large diversity of bacteriocins known and most target very specific microbial populations. Thus, a microbial product that contains multiple strains may be able to produce multiple bacteriocins and target different groups of pathogenic populations. Conversely, the intestinal tract contains a large diversity of bacteriocin producing bacteria. While some of the produced bacteriocins may affect one of the included strains, it is unlikely to affect all of the included microorganisms.
Another benefit of a multiple-strain containing product is the ability to target more than one pathogen population. Microbial pathogens are very diverse are require different methods to reduce or eliminate their populations. Thus, a product containing different microorganisms that are able to effect different pathogenic populations will result in an overall healthier system.
Different microorganisms positively influence the gastrointestinal system through different mechanisms. Including bacteria that work through different methods may result in a superior product. One strain may reduce pathogen populations, while another has an immunostimulative effect, while another produces micronutrients essential for the host. Interestingly, multiple strains may also provide synergistic effects upon the host or pathogen inhibition abilities. One strain alone may not be able to reduce certain populations, but the combination of two different strains working through different mechanisms can reduce pathogen populations.
Additionally, the use of multiple beneficial microorganisms can help overcome bacteriophages that infect and kill bacteria. Bacteriophages are very common in gastrointestinal systems and have profound effects upon the microbial community. Bacteriophages require specific sites on a cell to bind and infect. Thus, including multiple microorganisms in a product, the greater the likelihood that at least some populations from the product will evade bacteriophage attack and elicit beneficial effects upon the microbial community and host.
Some research has illustrated that a combination of different strains of beneficial probiotic bacteria can be used to treat discrete disorders. For example US Pub. No. 20070280910 describes a probiotic composition that includes three different bacterial species consisting of Bacillus subtilis, Bacillus coagulans, and Enterococcus faecium purportedly to treat autism, yeast infections, fybromyalgia, and irritable bowel syndrome. However, what is needed is a combination of probiotic bacteria compositions for administration to animals to eliminate or reduce gastrointestinal pathogens where a single probiotic bacteria species may be resisted by evolving pathogenic bacteria or phages found in the gastrointestinal environment. The novel approach described here meets these needs by providing a microbial composition that will decrease the incidence of pathogens, decrease animal mortality, improve animal health, and maximize animal efficiency.